Thrombopoietin (TPO) is a hepatocyte-derived growth factor known to promote platelet number, to have growth-promoting potential for megakaryocytes (MKs), and to promote erythrocyte, monocyte, mast cell, and granulocyte proliferation in the presence of specific growth factors. To explore the ability of TPO to support mast cell proliferation, we cultured CD34+ cells in rhTPO under conditions developed to culture MKs and examined these cultures for the presence of HuMCs. We similarly added rhTPO to CD34+ cells cultured in stem cell factor (SCF), known to promote HuMC development. Using serum-free media (SFM) supplemented with rhTPO, human CD34+ cells at 2-3 weeks differentiated into MKs (85-90%) and (5-10%) poorly differentiated HuMC. SCF-independent HuMCs expressed TPO (c-Mpl) receptors, tryptase and chymase; and were capable of surviving in SFM with rhTPO alone, or proliferating when recultured in rhSCF. Small numbers of HuMC were noted when rhTPO was combined with 10 ng/ml rhSCF. However, in HuMC cultures with 100 ng/ml rhSCF, the addition of 50 ng/ml rhTPO significantly reduced HuMC numbers, with the appearance of MKs. Under all conditions, the addition of TPO favored the growth of KIT-low HuMC. In SCF dependent human mast cell cultures, we added both Th1 and TH2 cytokines and examined the effect of these cytokines (IL-4, IL-5, IFN-gamma)on mast cell numbers. We employed high resolution tracking of cell devision and correlated results with apoptosis, Kit expression, and mast cell degranulation. We found that all added cytokines decreased cell numbers, although the mechanisms differed. IFN-gamma decreased early progenitor cell division, IL-4 blocked Kit expression, and IL-5 blocked later cell division. Both IL-4 and IFN-gamma suppressed degranulation and the expression of the high affinity receptor for IgE. When these cytokines were administered to mature mast cells, IL-4 was unique in inducing some division and in enhancing degranulation. Such data is critical for anticipating the effects of cytokine manipulation in vivo on mast cell function.